Resveratrol Inhibits High Glucose-Induced H9c2 Cardiomyocyte Hypertrophy and Damage via RAGE-Dependent Inhibition of the NF-κB and TGF-β1/Smad3 Pathways

Figures

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  Figure 1. Effects of RES on HG-stimulated H9c2 cell viability. Cells were subjected to treatment with different concentrations (0–400 μg/mL) of RES for 24 h, then assayed with a CCK-8 kit. (a) Chemical structure of RES; (b) cell viability. The data are shown as the mean ± SD of the three repeated trials; ∗p < 0.05 vs. 0 μg/mL (RES) group.

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  Figure 2. RES's effects on HG-induced hypertrophy in H9c2 cells. Cells were subjected to treatment with control medium or HG containing 20 and 40 μg/mL RES for 24 h. The BCA method and WB analysis of the content of total protein content in H9c2 cells for ANP and β -MHC were performed. (a) Content of total protein content in H9c2 cells; (b) representative WB images of ANP, β -MHC, and GAPDH; (c) ANP'S relative protein expression; (d) β -MHC's relative protein expression. The levels of protein were relatively determined by densitometry and normalised to the level of GAPDH. The data are presented as the mean ± SD of the three repeated trials; ∗p < 0.05vs. HG group.

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  Figure 3. Effects of RES on cell apoptosis in HG-treated H9c2 cells. H9c2 cells were subjected to treatment with control medium or indicated concentrations of RES (20 and 40 μg/mL) for 24 h. Flow cytometry and WB analysis of cell apoptosis markers, Bax, Bcl‐2, cleaved caspase‐3, and GAPDH were conducted. (a) Cell apoptosis of H9c2 cells; (b) representative WB images of these markers; (c) ratio of Bax/Bcl‐2; (d) protein expression of cleaved caspase‐3. The levels of protein were determined by densitometry and normalised to the levels of GAPDH. The data are presented as the mean ± SD of the three repeated experiments; ∗p < 0.05vs. HG group.

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  Figure 4. RES's effects of on HG-stimulated activation of the RAGE/NF-κB and inflammatory cytokines production. H9c2 cells were subjected to treatment with control medium or suggested concentrations of RES (20 and 40 μg/mL) for 24 h. ELISA, RT-PCR, and WB analysis of IL-6, TNF-α, RAGE, p-NF-κB P65, P65, and GAPDH levels were performed. (a) IL-6's secretion level of IL-6 in supernatant; (b) TNF-α's secretion level in supernatant; (c) IL-6 and TNF-α's mRNA levels in H9c2 cells; (d) representative WB images of RAGE, p-P65, P65, and GAPDH; (e) RAGE's protein expression; (f) p-P65's protein expression. The levels of protein were determined by densitometry and normalised to the level of GAPDH. We normalised the relative mRNA levels to that of GAPDH. The data are presented as the mean ± SD of the three repeated trials; ∗p < 0.05vs. HG group.

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  Figure 5. Effects of RES on activation of the TGF‐β1/Smad3 pathway and ECM components induced by HG. H9c2 cells were subjected to treatment with control medium or suggested RES concentrations (20 and 40 μg/mL) for 24 h. WB analysis of TGF‐β1, p-Smad3, Smad3, collagen 1, α-SMA, and GAPDH levels was performed. (a, d) Representative WB images of TGF‐β1, p-Smad3, Smad3, collagen 1, α-SMA, and GAPDH levels; (b) protein expression of TGF‐β1, (c) p-Smad3, (e) collagen 1, and (f) α-SMA. The levels of protein were determined by densitometry and normalised to the levels of GAPDH. We normalised the relative mRNA levels to the levels of GAPDH. The data are presented as the mean ± SD of the three repeated trials; ∗p < 0.05vs HG group.

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  Figure 6. RES suppressed the HG‐induced activation of pathways of TGF‐β1/Smad3 and NF-κB dependent on RAGE in vitro. H9c2 cells were treated with control medium or indicated concentration of RES (40 μg/mL) or FPS-ZM1 (100 μM) for 24 h. WB analysis of RAGE, p-NF-κB P65, P65, TGF‐β1, p-Smad3, Smad3, and GAPDH levels was performed. (a, d) Representative WB images of RAGE, p-NF-κB P65, P65, TGF‐β1, p-Smad3, Smad3, and GAPDH levels; (b) protein expression of RAGE, (c) p-NF-κB P65, (e) TGF‐β1, and (f) p-Smad3. The levels of protein were determined by densitometry and normalised to the levels of GAPDH. The data are presented as the means ± SD of the three repeated experiments; ∗p < 0.05 vs, HG group.

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  Figure 7. Schematic drawing of the cardioprotective effects of resveratrol on HG‐stimulated H9c2 cardiomyocyte damage.